EP0654532B1 - Antimucoglycoprotein monoclonal antibody - Google Patents
Antimucoglycoprotein monoclonal antibody Download PDFInfo
- Publication number
- EP0654532B1 EP0654532B1 EP94916400A EP94916400A EP0654532B1 EP 0654532 B1 EP0654532 B1 EP 0654532B1 EP 94916400 A EP94916400 A EP 94916400A EP 94916400 A EP94916400 A EP 94916400A EP 0654532 B1 EP0654532 B1 EP 0654532B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- monoclonal antibody
- mucus
- pbs
- cells
- washed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
- G01N2474/20—Immunohistochemistry assay
Definitions
- the Concanavalin A paradoxical staining cannot quantitatively determine the gastric gland-type mucus because of its poor reproducibility of staining and also because of the drawback that the method can be applied only to fixed specimens and hence cannot be applied to the determination of secreted or solubilized mucus.
- the culture supernatant is sampled from each of the wells with respect to which hybridoma has been confirmed to be formed by the cultivation in D. above, and assayed for the presence of immunoreactivity with gastric mucus glycoprotein.
- screening may preferably be performed by ELISA, for example.
- Immunoreaction-positive samples are further screened for specific recognition of gastric gland mucous cells and the mucus secreted thereby.
- Such screening may preferably be carried out, for example, by immunohistochemical staining using sections of fixed gastric mucosa specimens.
- the thus purified monoclonal antibody may be labeled by any biochemical means widely used in the art with peroxidase, alkaline phosphatase, biotin etc. Labeling may be performed by linking product from periodate oxidation of the enzyme to the antibody or by combining the enzyme and antibody together using glutaraldehyde as crosslinking agent.
- the thus obtained labeled monoclonal antibody can be conveniently applied to immunohistochemical staining or ELISA-based sandwich assay.
- the microplate After incubation at room temperature for 30 minutes, the microplate was visually observed to find that the only two wells pretreated with the monoclonal rat anti-mouse IgM reagent and the monoclonal rat anti-mouse IgL (K) reagent, respectively, showed an intense yellow coloration, the other wells being colorless and transparent.
- the monoclonal antibody of the present invention was identified as an IgM antibody with the light chain being a ⁇ chain.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
Centrifugation | 1.5 x 105 g |
Temperature | 10°C |
Period | 85 hours |
Apparatus | Model 72P; Rotor : RPS-40T (Hitachi, Ltd.) |
ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05 M sodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at a concentration of 2 µg/ml and the same buffer was used to prepare two-fold serial dilutions. 100 µl aliquots of each dilution was added to the wells of a microplate adapted for use in ELISA (Corning) and were allowed to stand overnight at 4°C. Each well was washed three times with 0.05% Tween 20 in PBS (PBS-Tween), filled with 2% skim milk in PBS, and then allowed to stand for 1 hour. The wells were washed three times with PBS-Tween and a 1,000-fold dilution of mouse serum sample was dispensed in 100 µl aliquots into the wells. After incubation for 1 hour, the wells were washed three times with PBS-Tween, and 100 µl of a 10,000-fold dilution in PBS of peroxidase-labeled goat anti-mouse immunoglobulins antibody (Tago, Inc.) was added as secondary antibody to each well and allowed to stand for 1 hour. The wells were washed three times with PBS-Tween and 100 µl of an ABTS-H2O2 peroxidase substrate solution (Kirkegaard & Perry Laboratories) was added to each well. After reaction at room temperature for 30 minutes, the optical density at 415 nm was measured for each well using a microplate reader. Those mouse sera which developed a color in dependence on the dose of the mucus glycoprotein were judged to be antibody-positive.
ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05 M sodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at a concentration of 1 µg/ml. 100 µl of the solution was added to each well of a microplate adapted for use in ELISA (Corning) and was allowed to stand overnight at 4°C. Each well was washed three times with 0.05% Tween 20 in PBS (PBS-Tween), filled with 2% skim milk in PBS and then allowed to stand for 1 hour. The wells were washed three times with PBS-Tween and 100 µl/well of hybridoma culture supernatant obtained in (5) above was added to each well. After incubation for 1 hour, the wells were washed three times with PBS-Tween, and 100 µl/well of a 10,000-fold dilution in PBS of peroxidase-conjugated goat anti-mouse immunoglobulins antibody (Tago, Inc.) was added as secondary antibody to each well and allowed to stand for 1 hour. The wells were washed three times with PBS-Tween and 100 µl of an ABTS-H2O2 peroxidase substrate solution (Kirkegaard & Perry Laboratories) was added to each well. After reaction at room temperature for 30 minutes, the optical density at 415 nm was measured for each well using a microplate reader. Those wells were selected which showed a more intense color development than the wells treated in the same manner as described above except that the myeloma cell culture supernatant obtained in (3) above was added instead of the hybridoma culture supernatant. The samples corresponding to the so selected color-developing wells were judged to be positive.
Immunohistochemical staining: Human gastric mucosa was fixed with formalin, embedded in paraffin and sliced into 4 µm-thick sections using a microtome. Each of the thus prepared sections was fixed on a slide glass. Each slide glass was dipped in xylene for deparaffinization and then in methanol containing 0.3% hydrogen peroxide for 30 minutes and then washed by dipping in PBS for 30 minutes. Subsequently, 10% rabbit normal serum (Nichirei Corporation) was applied to the thus washed section on each slide glass. After incubation for 1 hour, the sections were washed in PBS. Samples of hybridoma culture supernatant were applied onto the washed sections. After incubation for 1 hour, the sections were washed in PBS. Biotin-labeled rabbit anti-mouse IgG + IgA + IgM (H + L) antibody (Nichirei Corporation) was applied onto each washed section. After incubation for 1 hour, the sections were washed in PBS. Peroxidase-conjugated streptavidin (Nichirei Corporation) was then applied onto each washed section. After incubation for 1 hour, the sections were washed in PBS. Subsequently, each washed section was dipped for about 4 minutes in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.02% diaminobenzidine (Dojindo Laboratories) and 0.005% aqueous hydrogen peroxide, for color development.
Gastric mucosa | Corpus | Mucus gel layer | + |
Surface mucous cell | - | ||
Mucous neck cell | + | ||
Antrum | Mucus gel layer | + | |
Surface mucous cell | - | ||
Pyloric gland cell | + | ||
Duodenal mucosa | Villus epithelium | - | |
Brunner's gland | + | ||
N.B. +: positive to staining; -: negative to staining |
Competitive ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05 M sodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at a concentration of 2 µg/ml. 100 µl of the solution was added to each well of a microplate adapted for use in ELISA (Corning) and was allowed to stand overnight at 4°C. Each well was washed three times with 0.05% Tween 20 in PBS (PBS-Tween), filled with 2% skim milk in PBS and then allowed to stand for 1 hour. The wells were washed three times with PBS-Tween. In a separate container, 50 µl of the sample, i.e. the above-mentioned column eluate of mucus glycoprotein antigen decomposition product, 25 µl of culture supernatant from a 3-day culture of hybridoma HIK-1083 (Accession number P-13622) in normal medium [RPMI 1640 (Nissui Pharmaceutical Co., Ltd.) (10.2 g/l) medium supplemented with sodium hydrogen carbonate (2.2 g/l), L-glutamine (0.3 g/l), gentamycin (40 mg/l) and fetal calf serum (10% V/V)] and 25 µl of a 4-fold concentrate of PBS were mixed together and the mixture was allowed to stand for 2 hours. These mixture were added to the washed wells. After incubation for 1 hour, the wells were washed three times with PBS-Tween, and 100 µl of a 1:10,000 dilution in PBS of peroxidase-conjugated goat anti-mouse immunoglobulins antibody (Tago, Inc.) was added as secondary antibody to each well. After 1 hour of incubation, the wells were washed three times with PBS-Tween and 100 µl of an ABTS-H2O2 peroxidase substrate solution (Kirkegaard & Perry Laboratories) was added to each well. After 30 minutes of incubation at room temperature, the optical density at 415 nm was measured for each well using a microplate reader. Those samples corresponding to the wells which gave lower optical density than that of the reaction solution in the well where the above-described procedure was performed using distilled water instead of the samples were judged as ones having antigenic activity.
Competitive ELISA: The purified mucus glycoprotein antigen was dissolved in 0.05 M sodium carbonate-sodium hydrogen carbonate buffer, pH 9.6, at a concentration of 2 µg/ml. 100 µl of the solution was added to each well of a microplate adapted for use in ELISA (Corning) and was allowed to stand overnight at 4°C. Each well was washed three times with 0.05% Tween 20 in PBS (PBS-Tween), filled with 2% skim milk in PBS and then allowed to stand for 1 hour. The wells were washed three times with PBS-Tween. In a separate container, 50 µl of the sample, i.e. the above described aqueous solution, 25 µl of culture supernatant from a 3-day culture of hybridoma HIK-1083 (Accession number P-13622) in normal medium [RPMI 1640 (Nissui Pharmaceutical Co., Ltd.) (10.2 g/l) medium supplemented with sodium hydrogen carbonate (2.2 g/l), L-glutamine (0.3 g/l), gentamycin (40 mg/l) and fetal calf serum (10% V/V)] and 25 µl of 4-fold concentrate of PBS were mixed together and the mixture was allowed to stand for 2 hours. These mixtures were added to the washed wells. After incubation for 1 hour, the wells were washed three times with PBS-Tween, and 100 µl of a 1:10,000 dilution in PBS of peroxidase-conjugated goat anti-mouse immunoglobulins antibody (Tago, Inc.) was added as secondary antibody to each well. After 1 hour of incubation, the wells were washed three times with PBS-Tween and 100 µl of an ABTS-H2O2 peroxidase substrate solution (Kirkegaard & Perry Laboratories) was added to each well. After 30 minutes of incubation at room temperature, the optical density at 415 nm was measured for each well using a microplate reader.
Claims (5)
- An IgM class monoclonal antibody which recognizes sugar chains comprising α -linked N-acetylglucosamine residues, and which specifically reacts with mucus glycoproteins produced by human gastric gland-type mucous cells.
- The monoclonal antibody of claim 1, wherein the IgM class monoclonal antibody is obtainable by a hybridoma deposited in the National Institute of Bioscience and Human-Technology (NIBH), Agency of Industrial Science and Technology with the accession number P-13622.
- A hybridoma which produces an IgM class monoclonal antibody according to claim 1.
- A hybridoma deposited in the National Institute of Bioscience and Human-Technology (NIBH), Agency of Industrial Science and Technology with the accession number P-13622.
- Use of the monoclonal antibody of claim 1 or 2 for the analysis of gastric gland-type mucous cell-derived mucus glycoproteins contained in human body fluids.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP15989193 | 1993-05-26 | ||
JP159891/93 | 1993-05-26 | ||
JP15989193A JP3581160B2 (en) | 1993-05-26 | 1993-05-26 | Anti-mucus glycoprotein monoclonal antibody |
PCT/JP1994/000838 WO1994028158A1 (en) | 1993-05-26 | 1994-05-26 | Antimucoglycoprotein monoclonal antibody |
Publications (3)
Publication Number | Publication Date |
---|---|
EP0654532A1 EP0654532A1 (en) | 1995-05-24 |
EP0654532A4 EP0654532A4 (en) | 1996-04-24 |
EP0654532B1 true EP0654532B1 (en) | 2001-08-01 |
Family
ID=15703440
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP94916400A Expired - Lifetime EP0654532B1 (en) | 1993-05-26 | 1994-05-26 | Antimucoglycoprotein monoclonal antibody |
Country Status (6)
Country | Link |
---|---|
US (1) | US5576182A (en) |
EP (1) | EP0654532B1 (en) |
JP (1) | JP3581160B2 (en) |
CA (1) | CA2141025C (en) |
DE (1) | DE69427850T2 (en) |
WO (1) | WO1994028158A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5928433A (en) * | 1997-10-14 | 1999-07-27 | The Lubrizol Corporation | Surfactant-assisted soil remediation |
US20150231077A1 (en) | 2014-02-17 | 2015-08-20 | The Cleveland Clinic Foundation | Amine passivated nanoparticles for cancer treatment and imaging |
CN113899896A (en) * | 2021-08-16 | 2022-01-07 | 南京理工大学 | Microchip for identifying selenium sugar, qualitative analysis method of selenium sugar and application |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5183756A (en) * | 1988-08-19 | 1993-02-02 | The United States Of America As Represented By The Department Of Health And Human Services | Monoclonal antibody (D612) having selective reactivity for gastrointestinal caricinomas and method for employing the same |
-
1993
- 1993-05-26 JP JP15989193A patent/JP3581160B2/en not_active Expired - Fee Related
-
1994
- 1994-05-26 CA CA002141025A patent/CA2141025C/en not_active Expired - Fee Related
- 1994-05-26 DE DE69427850T patent/DE69427850T2/en not_active Expired - Fee Related
- 1994-05-26 US US08/373,220 patent/US5576182A/en not_active Expired - Lifetime
- 1994-05-26 EP EP94916400A patent/EP0654532B1/en not_active Expired - Lifetime
- 1994-05-26 WO PCT/JP1994/000838 patent/WO1994028158A1/en active IP Right Grant
Non-Patent Citations (2)
Title |
---|
Biochem. J., 318, 1996, 409-416 * |
J. Histochem. Cytochem., 46(7), 1998, 793-801 * |
Also Published As
Publication number | Publication date |
---|---|
JPH06335396A (en) | 1994-12-06 |
EP0654532A4 (en) | 1996-04-24 |
EP0654532A1 (en) | 1995-05-24 |
CA2141025C (en) | 2005-08-16 |
JP3581160B2 (en) | 2004-10-27 |
DE69427850T2 (en) | 2001-12-06 |
CA2141025A1 (en) | 1994-12-08 |
DE69427850D1 (en) | 2001-09-06 |
US5576182A (en) | 1996-11-19 |
WO1994028158A1 (en) | 1994-12-08 |
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